The invention relates to methods and test kits for diagnosis of periodontal disease activity in mammals, especially in human. The methods of the invention provide for rapid chair-side diagnosis of periodontitis, peri-implantitis and HIV (+)-infection/AIDS-disease related periodontal diseases. Especially, the methods of the invention provide for rapid chair-side diagnosis of the loss of bone density associated with periodontal diseases.
Periodontal diseases are a major problem in the human dentition. In fact, more teeth are lost from periodontal disease than from dental caries. Thus, there is a great need for reliable diagnostic tests for periodontal disease.
Periodontal disease comprises a group of inflammatory disorders originating from infections affecting the gingiva (gum), periodontal ligament (a periodontal structural element/tissue linking tooth to alveolar bone) and the alveolar (law) bone structures supporting the teeth. The primary cause of periodontal diseases is bacterial plaque attached to the teeth. This causes inflammation of the gum which may result in destruction of the actual tooth-supporting structure and bone. In periodontal disease, there is usually a large accumulation of bacteria in plaque, both above (supragingival) and below (subgingival) the gum line. The plaque can calcify and form calculus deposits. The calculus deposit and associated plaque can create a "pocket" between the teeth and the gingiva which is an irreversible characteristic of periodontal disease.
Gingivitis (gum inflammation) is distinguished from periodontitis in that in gingivitis, gingiva are inflamed but no deep (&gt;4 mm) periodontal pockets are detectable; thus, no irreversible destruction of tooth supporting structures is associated with gingivitis. Periodontitis is characterized by inflamed gingiva and destruction of tooth supporting structures; however, periodontitis can be missed in clinically-healthy-looking gingiva.
Several methods for detecting periodontal disease have been developed (Armitage, G. C., C.D.A. Journal 36, 35-41, 1993). However, none of the presently available detection methods is sufficiently accurate and specific to provide a reliable tool for diagnosing and assessing the hard tissue destruction associated with periodontal diseases, including peri-implantitis and HIV (+)-infections/AIDS-related periodontal diseases.
Especially, several attempts to develop methods for assessing progressing periodontitis, as discussed below, have been tested but none of them have been found to be satisfactory enough to create a rapid and reliable chair-side test for the hard tissue destruction (bone resorption) associated with periodontal disease.
Visual Examination
When gingiva (gums) are affected by periodontal disease, color change (from pink to red), texture alterations (redness and swelling), and an increased tendency to bleed (in that particular gingival and sulcular/sulcus area) can be detected. Advanced stage periodontal disease is frequently associated with increased tooth mobility and drifting of the teeth.
However, some forms of periodontal disease, such as localized juvenile periodontitis (LJP), can have a treacherous nature and a misleading clinical course. Thus, active local periodontitis is not always detectable by visual examinations. Consequently, biochemical adjunctive means to help the clinical diagnosis of juvenile periodontitis would be desirable and helpful for prompt and adequate early diagnosis, as well as identification and screening, especially in case of young patients.
Clinical Assessment of Periodontal Status and Probing of Periodontal Lesions
Currently, periodontal disease is diagnosed by clinical observation of indicators such as presence and depths of periodontal pockets, loss of attachment of the teeth to the bone, and papillary bleeding of the gums. Clinical observations, however, are not always reliable indicators. For example, even deep periodontitis pockets containing putative periodontal pathogens are not necessarily indicative of disease activity or periodontal tissue destruction.
Periodontal attachment levels can be assessed by means of a graduated periodontal probe and expressed as the distance from the cement enamel junction to the bottom of the gingival pocket. The longer distance for each tooth surface is recorded and may be included in the periodontium chart. Pocket depth values &lt;4 mm are excluded from the chart as falling within normal variations. Thus, pockets &gt;4 mm are considered as periodontitis pockets or periodontitis lesions.
As a measurement technique, periodontal probing has several sources of error. The extent of probe penetration varies with insertion force, inflammatory status of the periodontal tissues, and diameter of probe tip. Measurement errors resulting from thickness of the probe, contour of the tooth surface and improper angulation of the probe can be reduced or avoided by the selection of a proper instrument and careful management of the examination procedure. More difficult to avoid, however, are errors resulting from variations in probing force and inflammation of the periodontal tissues. The measurement errors limit the accuracy and reproducibility.
Automated periodontal probes have also been developed. The primary advantage is controlled insertion force, reproducibility, and direct data entry. The main disadvantages include reduced tactile sense of operator and patient discomfort.
Noteworthy, gingivitis or gum inflammation is distinguished from periodontitis by the facts that in gingivitis, unlike periodontitis, gingiva is inflamed but no deep (&gt;4 mm) periodontal pockets can be detected; thus, no irreversible degradation (destruction) of tooth supporting structures either detected by probing and/or radiographically is associated with gingivitis. Periodontitis is characterized by inflamed gingiva and destruction of tooth supporting structures; however, periodontitis can well exist under "clinically-healthy-looking" gingiva.
In conclusion, it is clear that clinical observations are not always reliable indicators. A further problem is the difficulty to assess an progressing periodontal disease because in some cases deep periodontitis pockets--even harboring putative periodontopathogens--are not necessarily active in regard to the inflammatory periodontal tissue destruction.
Radiographic Evaluation
Sequential radiographic images have also been used to evaluate periodontal disease activity. The loss of bone density at the alveolar crest is frequently a sign of progression of periodontitis.
The height of the alveolar (jaw) bone and the outline of the bone crest can be examined in the radiographs. The radiographs provide information of the height and configuration of the interproximal alveolar bone. However, the radiographic assessment of the periodontal disease activity has drawbacks. Even with an excellent set of films and an experienced examiner, the unaided eye can only detect changes in bone after 30-50 percent of the bone mineral has been lost. Cover structures (bone, tissue, teeth) often make it difficult to properly identify the outlines of buccal and lingual alveolar crests. The analysis of radiographs is to be combined with a detailed evaluation of the pocket depths and the attachment level data to obtain a correct and exact diagnosis. Upon recalls (examinations of treated periodontitis patients) radiographic examination is required.
In summary, periodontal probes and radiographs measure two separate components in the progression of periodontitis. One provides an estimate of the attachment loss of soft tissue from the tooth surface and the other measures loss of bone density.
Biological Tests
In addition to periodontal probing, biological (microbial and biochemical) tests have been designed to provide information associated with progressing periodontal lesions. These biological periodontitis tests fall into four general categories and are designed to detect the presence of 1) substances associated with putative pathogens, 2) tissue breakdown products, 3) proinflammatory and immunological mediators, and 4) host-derived proteins, enzymes and substances.
Biochemical marker research of periodontal disease activity has focused its attention either on gingival crevicular fluid (GCF), peri-implant sucular fluid (PISF) or on saliva/mouthrinse samples. GCF is an inflammatory exudate that flows from inflamed human gingiva via the periodontal pocket to saliva. The degradative processes in diseased inflamed human gingival tissues are reflected in adjacent GCF and PISF, respectively. In clinical practice, GCF is easily collected by placing filter paper strips at the periodontal pocket orifice. Similarly, PISF is collected from the peri-implant orifice. Thus, GCF and PISF are attractive sources of potential markers for the progression of periodontitis. The advantage of GCF and PISF analysis is the site-specificity in regard to non-affected and affected sites. The disadvantage is in small sample volumes and technical difficulties in sample collection. On the other hand, in saliva/mouth-rinse samples the concentrations of the inflammatory mediators/enzymes are often diluted and do not reflect site specificity.
Tests for Presence of Putative Periodontal Pathogens
Much work has been done to determine which microorganism(s) are associated with progressing periodontitis (Armitage, G. C., Periodontal diagnostic aids, C.D.A. Journal 36, 35-41, 1993). In untreated patients with periodontitis, for example, the following bacteria (alone or in various combinations) have been suggested as putative pathogens: spirochetes (such as Treponema denticola), Porphyromonas (Bacteroides) gingivalis, Bacteroides forsythus, Prevotella (Bacteroides) intermedio, Campylobacter rectus (Wolinella recta), Eikenella corrodens, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Capnocytophaga sputigena, Peptostreptococcus micros, Streptococcus mitis. Selenomonas sp., Eubacterium sp. and Haemophilus sp. etc. These organisms have been identified in subgingival plaque samples by cultural analysis, microscopic examination, and by DNA probe analysis (Armitage, G. C., C.D.A. Journal 36, 35-41, 1993).
The BANA/BAPNA-test, based on hydrolysis of benzoyl-arginine-naphtylamide/benzoyl-arginine-p-nitroanilide, has identified the same putative pathogens (Armitage, C. G., C.D.A. Journal 36, 35-41, 1993). Since it is not known with certainty which of these organisms (if any) are responsible for the progression of periodontitis, their presence may not reflect actual periodontal disease activity.
Measurement of Tissue Breakdown Products
One of the major features of periodontitis is the destruction of the extracellular matrix (i.e. collagen) of the periodontium. Type I and III collagens are the predominant collagen types present in periodontium.
Increased concentrations of hydroxyproline and glycosaminglycans have been shown in gingival crevicular fluid of periodontitis patients. Specific N- and C-terminal collagen telopeptides have been studied in periodontitis gingival crevicular fluid. These collagen type I and III propeptides in gingival crevicular fluid are suggested to reflect both gingival collagen synthesis and degradation (Talonpoika, J., Ann. Univ. Turku, Serie D 142, 1994). However, instead of reflecting the gingival collagen degradation they may reflect more efficient collagen synthesis/turnover in general, and have been valuable when monitoring periodontal healing after periodontal treatment rather than actual periodontal destruction associated with active periodontal disease progression (Talonpoika, J., et al., J. Clin. Periodontol, 21. 320-333, 1994).
An indicator of bone resorption is the measurement of collagen breakdown fragments, containing the pyridinoline intermolecular crosslinks (these are relatively unique to calcified tissues), which are released into biologic fluids (e.g., GCF, urine, serum) during extracellular destruction of bone (and cartilage). The measurement of these pyridinoline-containing collagen fragments in serum and urine is currently used as a diagnostic "marker" of active bone resorption during metabolic bone diseases such as hyperparathyroidism, post-menopausal osteoporosis, Paget's disease, and the arthritides (Greenwald R A, Arth Rheumat 39:1455-1465, 1996; Garnero P, Grimaux M, Sequin P, and Delmas P D, J Bone Min Res 9:255-264, 1994.) Giannobile et al. adapted the serum assay developed by Risteli et al. (1993), to monitor alveolar bone loss by measuring pyridinoline crosslinked carboxyterminal telopeptide fragments of type I collagen (ICTP) in GCF of periodontal pockets (Giannobile W V, Lynch S E, Denmark R G, Paquette D W, Fiorellini J P, and Williams R C, J Clin Periodontol 22:903-910, 1995; Giannobile W V, Palys M, Howell T H E, Haffajee A D, and Socransky S S, J Dent Res 75 (Spec. Issue):IADR abstract #1114 1996). Longitudinal studies on experimentally-induced periodontitis in dogs demonstrated that the detection of elevated levels of pyridinoline-containing fragments of type I collagen (this type of collagen makes up over 90% of the organic matrix of bone) in GCF preceded the detection of osseous metabolic activity and bone loss assessed by measuring bone-seeking radiopharmaceutical uptake and subtraction radiography, respectively (Giannobile W V, et al., J Clin Periodontal 22:903-910, 1995.). These pyridinoline-containing type I collagen fragments in GCF have also been reported to be positively correlated with clinical parameters of periodontal disease severity in humans, including radiologic assessment of alveolar bone loss, and were found to be decreased by conventional treatment such as scaling and root planing (Giannobile W V et al., J Dent Res 75 (Spec Issue):IADR abstract#1114, 1996, Talonpoika J T et al., J Clin Periodontol 21:320-326 1994). However, these assays do not provide a reliable, simple and rapid chair-side test. On the contrary, these assays are time-consuming and require expensive equipment. Moreover, such assays lack specificity since they may reflect the breakdown of collagen from inflamed tissue as well as from bone.
Measurement of Proinflammatory and Immunological Mediators
Periodontal tissues and especially some distinct cells connected with gingivitis and periodontitis are known to produce a variety of proinflammatory and immunological mediators. Some of these have been suggested as biochemical/immunological markers in the assessment of periodontal disease activity (Page, R. C., J. Periodont. Res. 26, 533-546, 1991). Increased amounts of these proinflanmmatory mediators are detected in diseased gingival tissue and gingival crevicular fluid relative to periodontally healthy gingiva and gingival crevicular fluid (Page, R. C., J. Periodont. Res. 26, 533-546, 1991). Tumor necrosis factor-.alpha., interleukin-1 .beta. and prostaglandin E2 have been subject of research (Page, R. C., J. Periodont. Res. 26, 533-546, 1991). As these mediators reflect the activity of the inflammatory process, they show promise as markers. Moreover, it has been observed that these bone-modulating cytokines (IL-1.beta. and TNF-.alpha.) were elevated in GCF at sites undergoing orthodontically-induced bone resorption, but not at inactive sites (Uematsu S, Mogi M, Deguchi T, J Dent Res 75:562-567, 1996). Yet none of them has proven to be specific enough to periodontal disease. Moreover, rapid tests for the detection of these bone modulating cytokines has not been developed.
In vitro these proinflammatory mediators are known to be capable of inducing de-novo MMP-expression by resident oral cells (gingival fibroblasts and keratinocytes) (Birkedal-Hansen, H. J. Periodontol. 64, 474-484, 1993). In vivo, increased amounts of the inflammatory mediators in periodontitis gingiva and gingival crevicular fluid are not associated with increased amounts of fibroblast-type MMPs (MMP-1 and MMP-2). These types of MMPs, in contrast to the PMN-type MMPs, are expressed and produced by resident gingival/oral fibroblasts and epithelial cells. Neutrophil-derived MMPs and elastase are found in periodontitis gingiva, gingival crevicular fluid and salivary/mouthrinse samples (Suomalainen, K., Thesis, Univ. Helsinki, 1993; Inigman, T., Thesis. Univ. Helsinki, Finland, 1994). Therefore, the relationships of the inflammation mediators (tumor necrosis factor-.alpha., interleukin-1-.beta. and prostaglandin E2 etc.) to MMP-dependent periodontal tissue destruction despite promising in vitro results (Birkedal-Hansen, H., J. Periodontal. 64, 474-484, 1993) is unclear (Sorsa, T., et al., Ann. N.Y. Acad. Sci. 732, 112-131, 1994).
Tests Based on Host-derived Proteins, Enzymes and Substances
The tissue destruction associated with the progression of periodontal disease/periodontitis lesions could be due to the independent and cooperative action of various host and bacterial derived proteolytic enzymes. During the development and progression of periodontitis lesions, various enzymes including matrix metalloproteinases (MMPs), elastase, cathepsin and trypsin-like proteinases etc. are released from triggered host cells. To some extent the proteinases may also be derived from oral bacteria (Uitto V-J, et al., Proc. Finn, Dent. Soc., 83, 119-130, 1987). Therefore, several proteinases, proteases and enzymes of this type have been suggested as biochemical markers for monitoring the progression and activity of periodontal disease (Armitage, C. G., C.D.A. Journal, 36, 35-41, 1993).
Test methods based on identification and measurement of enzymes/proteins and their activities have been developed. For example, aspartate aminotransferase (ASAT) has been associated with periodontal disease. This enzyme has been measured in gingival crevicular fluid, an inflammatory exudate of adjacent gingiva known to reflect the actual gingival cell tissue health (Page, R. C., J. Periodont, Res. 26, 230-242, 1991). ASAT is, however, released by almost all damaged cells in various periodontal tissues and is also present in blood in significant amounts. Therefore, a gingival crevicular fluid ASAT test is hampered by high non-specific background.
False positives caused by enzymes released in conditions other than active periodontal disease are a major problem when using most of the other suggested enzymes (e.g. .beta.-glucuronidase, lactate dehydrogenase, arylsulfatase, and some proteinases). Chair-side tests have been developed for gingival fluid elastase (Prognostick, Dentsply Corp., York, Pa.) and general proteinase activities (Periocheck.RTM., Advanced Clinical Technologies Inc., Westword, Mass.). Both these tests lack specificity. The synthetic peptides and gelatin used as substrates are degraded by almost all human and bacterial proteinases. Therefore, high background activities, false positive, and false negative results have been found when enzyme activity is correlated with the clinical course of periodontal disease as compared with periodontally healthy controls.
A chair-side calorimetric protease assay system has been suggested for diagnosing periodontal diseases from saliva/mouthrinse, gingival crevicular fluid and dental plaque samples (Periocheck.RTM., Advanced Clinical Technologies Inc., Westword, Mass.) has been developed.
The test known as the BANA/BAPNA-test, is based on hydrolysis of benzoyl-arginine-naphtylamide/benzoyl-arginine-p-nitroanilide. Hydrolysis results in red-orange color that indicates the presence of anaerobic oral bacteria such as Porphyromonas gingivalis, Treponema denticola and Bacteroides forsythus (Armitage, C. G., C.D.A. Journal, 36, 35-41, 1993). These organisms are thought to release trypsin-like proteases capable of hydrolyzing the synthetic BANA/BAPNA-peptide substrates. However, this test is not specific solely to bacterial proteinases. Many host cell derived proteinases (such as trypsins, trypsin-like proteinases, mast cell tryptases etc.) have similar specificities for BANA/BAPNA-peptides (Ingman, T., et al., Oral Microbiol, Inmunol, 8, 298-305, 1993). Human cell-derived trypsin-like proteinases in periodontitis gingival crevicular fluid have been shown (Sorsa, T., et al., J. Dent. Res. 71, 732, 1992). Thus, a problem with this test is that it does not distinguish between host-cell derived and bacterial proteinases.
In summary, in the above-described assays, because of the involvement of various host and bacterial derived proteinases in the degradation of non-specific synthetic or natural substrates, such as gelatin, false negative and positive results often occur.
Matrix Metalloproteinases (MMPs) in Periodontal Diseases
Enzymatic degradation of periodontal connective tissue accounts for various alternations that are characteristic of diseased periodontal tissues. These include the net reduction of collagen(s), decreased strength and increased permeability of periodontal/gingival tissue, and alveolar bone loss.
Loss of collagen can result from changes in collagen metabolism. The rate of synthesis by fibroblasts may be decreased in inflamed tissue; the collagen synthesized may include defects in molecular structure of fiber formation. These changes would render collagen more susceptible to proteolytic degradation. It has been shown that polymeric collagen fibrils containing intermolecular crosslinks are considerably more resistant to proteolytic/collagenolytic degradation than are soluble collagen fibrils.
Callagenolytic enzymes (collagenases, gelatinases and stromelysins, members of the matrix metalloproteinase (MMP) family) are a host cell-derived proteinase group that has been thoroughly studied in the context of periodontal disease. In culture conditions, explants of inflamed gingiva secrete more collagenase than do explants from clinically healthy gingiva. A number of lines of in vivo evidence implicate host cell-derived matrix metalloproteinases (MMPs) in human periodontal tissue destruction (Birkedal-Hansen, H., J. Periodontal. 64, 474-484, 1993). Evidence includes elevated collagenase activity (MMP-1 and MMP-8) and gelatinases (MMP-2 and MMP-9) in extracts of inflamed gingival tissues, gingival crevicular fluid and salivary/mouthrinse-samples of periodontitis patients (Sorsa, T., et al., Ann. N.Y. Acad. Sci., 732, 112-131, 1994). The activities of these proteinases have been found to be positively correlated with the severity of periodontal inflammation and pocket depth at the periodontitis lesion sites donating these proteinases to gingival extracellular matrix and adjacent gingiva (Sorsa, T., et al., Ann. N.Y. Acad. Sci., 732: 112-131, 1994). However, no statistically significant correlation has been found of actual loss of bone density associated with periodontal diseases and these proteinases.
The relative amount of these proteinases recovered in active rather than latent form appears to increase with the greater severity of the periodontal disease (Suomalainen, K., Thesis, Univ. Helsinki, Finland, 1993). Further, the activities of these MMPs in periodontitis sites decrease after instrumentation therapy (scaling and root planing) (Ingman, T., Thesis, Univ. Helsinki, Finland, 1994). Further, increased collagenase activity has been found in gingival crevicular fluid during experimental gingivitis (Sodek, J., et al., Matrix 12 (Suppl. 1), 352-362, 1992). Finally, more collagenase can be extracted from inflamed human gingiva than from less inflamed gingiva (Sorsa, T., Thesis. Univ. Helsinki, Finland, 1989).
These results are thought to reflect changes in mammalian MMP activity because the proteinases recovered from diseased sites degrade triple helical collagen into the 3/4- and 1/4-fragments that are characteristic of mammalian collagenase cleavage (Sorsa, T., Thesis, Univ. Helsinki, Finland, 1989; Sodek, J., et al., Matrix 12 (Suppl. 1), 352-362, 1992). Recent studies on collagenases in gingivitis/periodontitis in gingiva, gingival crevicular fluid, and saliva/mouthrinse samples have utilized the difference in cleavage patterns of collagen by vertebrate and bacterial collagenase to identify the origin of the collagenases (Sorsa, T., et al., Ann. N.Y. Acad. Sci., 732; 112-131, 1994). For example, collagen type I was incubated with gingival extracts of inflamed human gingiva, gingival crevicular fluid (GCF) and peri-implant sucular fluid (PISF) samples; then, the reaction products were analyzed. The results consistently showed a cleavage pattern characterized by human rather than bacterial collagenase. Thus, collagenase of gingival tissue, GCF and PISF samples has been proven to originate mainly from human cells and not from bacteria (Sorsa, T., et al., Ann. N.Y. Acad. Sci., 732: 112-131, 1994). Recent studies have also shown that salivary and dental plaque collagenases from human supragingival and subgingival dental plaque samples are of human origin. It has been suggested that these collagenases have functional and immunological characteristics of MMP-8 in active form (Sorsa, T., et al., J. Clin. Periodontal., 22: 709-717, 1995). Thus, it appears that collagenase in periodontal disease is derived mainly from the host.
Further characterization of gingival tissue, gingival crevicular fluid, and salivary/mouthrinse collagenases MMPs have revealed that the predominant source of the enzymes are polymorphonuclear neutrophilic leucocytes (PMN) present in periodontal inflammation. This is based on studies of the substrate specificity against type I-III collagens, response to procollagenase activators, and Western-blot and immunochemical analysis using specific anti-MMP antibodies (Sorsa, T., et al., Ann. N.Y. Acad. Sci. 732, 112-131, 1994). Tonetti, M. S., et al., have recently noticed that transcripts of neutrophil collagenase (MMP-8) can be found in inflamed human gingival tissue (Tonetti, M. S., et al., J. Periodont. Res. 28, 511-513, 1993).
If measured as total gingival crevicular fluid (GCF) and salivary collagenase or gelatinase activities, these proteinases have been found to be positively correlated with inflamed gingival tissue collagenase and gelatinase activities (Kinane, Curr. Op. Dent. 2, 25-32, 1992). Periodontal treatment results in decreased gingival crevicular fluid, salivary/mouthrinse PMN MMP-activities, close to those detected in healthy oral fluid samples (Sorsa. T. Thesis, Univ. Helsinki 1989; Suomalainen, K. Thesis, Univ. Helsinki, Finland, 1993; and Ingman, T. Thesis Univ. Helsinki, Finland, 1994).
Additionally, several studies have recently demonstrated that specially-formulated, low-dose regimens of doxycycline (LDD) can serve as matrix metalloproteinase inhibitors by suppressing the collagenase activity in the gingival crevicular fluid (GCF) and gingival tissues of patients with adult periodontitis (AP) (Golub, L M, et al., J Amer Dent Assoc 125:163-9 (1994), Golub, L M et al., J Periodontal Res 25:321-30 (1990)). In two subsequent placebo-controlled, double-blind clinical trials (one a multi-center study of 12 months duration), this regimen was also found to reduce pocket depth, improve periodontal attachment levels and inhibit alveolar bone loss in such patients (Crout, R J et al., J Periodontal 67:506-14 (1996), Caton, B J et al., J Dent Res 76 (Spec Issue):IADR abstract #1307 (1997)). Since LDD is an effective therapy for periodontal disease, the correlation between biochemical indicators, such as the MMPs, and the severity of periodontal disease can be assessed.
Several methods have been described to measure collagenolytic enzyme activity in saliva or gingival crevicular fluid. Activity can be measured spectrophotometrically by observing the increase in absorbance caused by collagen degradation (227 nm) (Lindy, S., et al., Eur. J. Biochem. 158, 1-4, 1986). Also, the degradation of a synthetic peptide as substrate connected to a color or fluorescence forming system can be followed spectrophotometrically or correspondingly, fluorometrically (Tschesche, H. et al., In Methods in Enzymatic Analysis, Bergmeyer, U. H. ed., Verlag Chemic, Weinhein, Germany pp. 239-248, 1985). With these methods, differentiation between individual collagenases is possible and can be achieved by specific inhibitors and activators (Sorsa., T., et al., Thesis Univ. Helsinki, Finland, 1989).
Matrix Metalloproteinase-8 (MMP-8) in Periodontal and Peri-implant Diseases
MMP-8 (also known as collagenase-2 or neutrophil collagenase) is produced as procollagenase (proMMP-8) by human polymorphonuclear neutrophilic leucocytes (PMNs). Sorsa T., et al. have suggesed that MMP-8 is the key member of the collagenase/ MMP-group. MMP-8 was said to be specifically involved in the progression of tissue destruction seen in periodontal disease. In fact, MMP-8 was suggested to be the primary MMP in the initiation of gingival/periodontal and alveolar bone tissue destruction in periodontal diseases. (Sorsa, T., et al., J. Periodont. Res. 23, 386-393, 1988; Sorsa, T., et al., Arch. Oral. Biol. 35, 193-6, 1990; Golub, L. M., et al., J. Clin. Periodont., 22, 100-109, 1995; Sorsa, T., et al., Ann. N.Y. Acad. Sci. 732, 112-131, 1994; Ingman, T., et al., J. Periodontol 64, 82-88, 1993). Also peridontitis associated with HIV (+) Infections/AIDS diseases (Robinson, P. G., et al., J. Periodont. 65, 236-243, 1994; Holmstrup, P., et al., J. Clin. Periodontol. 21, 270-280, 1994) was found to be associated with increased activities and amounts of MMP-8. (Salo., T., et al., Ann. N.Y. Acad. Sci., 732, 476-478, 1994). Also inflammatory processes associated with peri-implantitis were suggested be associated with the increased activities and levels of MMP-8 in peri-implantitis gingival crevicular fluid (Inginan, T., et al., J. Clin. Periodontol. 21, 301-307, 1994; Teronen, O., et al., J. Dent. Res. 76: 1527-1537, 1997).
MMP-8 from the GCF, PISF and salivary/mouthrinse samples of periodontitis and peri-implantits patients have been found to be converted from inactive, latent proforms to catalytically active forms by periodontal and peri-implant inflammation (Sorsa, T., et al., J. Peridont. Res. 23, 386-393, 1988; Uitto, V., et al., J. Periodont. Res. 25, 135-142, 1990). The activation of gingival crevicular fluid and salivary pro-MMs in periodontitis could result from independent and/or co-operative action of other human/PMN-proteinases (for example, cathepsin G, elastase), bacterial (P. gingivalis and T. denticola) proteinases and PMN-generated reactive oxygen species (such as hypochlorous acid. HOCI) (Sorsa, T., et al., N. Engl. Med. 321, 327-328, 1989; Sorsa, T., et al. Infection and Immunity 60, 4491-95, 1992; Sorsa, T., et al., Semin. Arth. Rheum. 22, 44-53, 1992).
A method which uses monoclonal antibodies which recognize the active mammalian MMP-8 has been disclosed (U.S. Pat. No. 5,736,341). The method is capable of differentiating between the active MMP-8 and its inactive proform. A chair-side test has been suggested for the detection of MMP-8 in the GCF, PISF or on saliva/mouthrinse samples by use of this method.
As will be shown below, however, the levels of MMP-8 in the GCF, PISF or saliva do not correlate well with the hard tissue destruction (bone loss) associated with periodontal diseases. MMP-8 is directly related instead to the soft tissue destruction (connective tissue destruction) that takes place during periodontal diseases.
As discussed above, a multitude of methods for assessing periodontal disease activities has been developed. However, none of these methods provide an adequate test to diagnose the bone destruction characteristic of periodontal diseases. The visual examination does not provide a diagnosis and an assessment of the progression of bone destruction. Clinical observations are not reliable enough because even deep pockets are not necessarily inflammatory active. Radiographic evaluations have to be combined with detailed clinical observations and visual examinations. The presence of pathogenic microorganisms do not fully reflect actual periodontic disease activity. Diagnoses based on breakdown products have not been satisfactory either because the presence of breakdown products may indicate rapid turnover or synthesis of collagen not necessarily degradation thereof. Proinflammatory mediators have been studied but no sufficiently rapid and specific test has been designed. Tests based on several host-derived enzymes have been developed, but most of them are not specific due to false positives caused by enzymes released by bacteria.
As mentioned above, although a rapid chair-side test which uses monoclonal antibodies has been suggested for MMP-8 (US Pat. No. 5,736,341), the levels of MMP-8 in the GCF, PISF or saliva are not good indicators of the bone loss associated with periodontal disease. MMP-8 is directly related, instead, to the soft tissue destruction and inflammation which takes place during periodontitis.
A biochemical marker test that detects periodontal disease activity in a simple, practical, and reliable manner requires sensitivity and specificity. Sensitivity is the probability that the disease is present when the test results are positive. Specificity is the probability that the disease is absent when the test results are negative. In the progression of periodontitis, an optimal test would detect all progressing periodontitis sites without registering false negative results (optimal sensitivity) and all nonprogressing sites without registering false-positive results (optimal specificity).
In view of the above considerations, it is clear that existing methods for diagonosing the hard tissue destruction associated with periodontal disease are limited in a number of ways. For example, the existing art does not provide a rapid, chair-side diagnostic test and assessment for the loss of bone density associated with periodontitis, peri-implantitis and HIV(+)-infection/AIDS-disease related periodontal diseases. The lack of a diagnostic test revealing and monitoring the bone resorption (hard tissue destruction) associated with periodontal disease activity has been a serious problem, particularly in view of the severity of the corrective measures typically required to be taken to treat the bone destruction of periodontal disease. Thus, it is important to find biochemical markers which correlate with bone resorption.
Accordingly, it is an object of this invention to overcome the above limitations presently encountered in the art, by providing a method of monitoring the course and treatment of the bone loss associated with periodontal disease with a rapid and reliable chair-side assay.